4.54.5 © Springer-Verlag Berlin Heidelberg 2005 II.4.5 Antiepileptics by Einosuke Tanaka Introduction As antiepileptics, phenytoin, mephenytoin, nirvanol and ethotoin are being used for treat- ments of grand mal and complex partial seizures. A iminostilbene derivative carbamazepine, phenobarbital (one of the barbiturates) and some benzodiazepines are also being well used as antiepileptics ( > Figure 5.1). Poisoning cases due to accidental and suicidal ingestion of these antiepileptics were reported [1]. For analysis of antiepileptics, methods using HPLC [2–15], LC/MS [16], GC [17, 18] and GC/MS [19,20] were reported. Among them, HPLC methods are being used most commonly. In this chapter, two HPLC methods, dealing with analysis of phenytoin and other antiepileptics together with their metabolites in serum [11] and plasma [15], are presented. ⊡ Figure 5.1 Structures of antiepileptics and their analogs. 352 Antiepileptics HPLC analysis of antiepileptics in serum [11] Reagents and their preparation • Carbamazepine, ethosuximide, phenytoin, primidone, phenobarbital, diazepam, oxaze- pam, nitrazepam and 5-(4-methylphenyl)-5-phenylhydantoin (internal standard, IS) can be all purchased from Sigma (St. Louis, MO, USA). • Each of above drugs is dissolved in methanol to prepare 1 mg/mL solution. • By diluting the 1 mg/mL solution with methanol, solutions at various concentrations in the range of 0.05–5 µg/mL are prepared for drugs to be quantitated, because they are used for constructing each calibration curve. HPLC conditions Column: a reversed phase column a ( TSK gel super-ODS; 100 × 4.6 mm i. d., particle diameter 2 µm, Tohso, Tokyo, Japan). Mobile phase: acetonitrile/8 mM phosphoric acid aqueous solution (3:7, v/v); its  ow rate: 0.6 mL/min; detection wavelength: 215 nm; column (oven) temperature: room temperature. Procedure i. A 0.5-mL volume of serum b , 100 µL of 0.2 M hydrochloric acid solution, 50 µL of IS solu- tion and 3 mL dichloromethane are placed in a glass centrifuge tube with a ground-in stopper. ii. A er shaking for 1 min, the tube is centrifuged. iii.  e resulting organic phase (lower layer) is transferred to a glass vial with a conical bottom. iv.  e organic extract is evaporated to dryness under a stream of nitrogen. v.  e residue is dissolved in 100 µL mobile phase. vi. A  xed amount of the above solution is injected into HPLC. vii. For constructing calibration curves, various concentrations of standard solution are mixed with 50 µL IS solution each and processed according to the above procedure. Assessment of the method > Figure 5.2 shows an HPLC chromatogram for the eight antiepileptics listed in > Figure 5.1, which had been spiked into human serum.  e linearity of each compound could be obtained in the range of 0.05–1 µg/mL in serum; recovery rates were 96–104 %.  e detection limits were: 0.05 µg/mL for carbamazepine; 0.1 µg/mL for primidone, phenobarbital, phenytoin c , oxazepam, ethosuximide, and nitrazepam; and 0.5 µg/mL for diazepam. 353 HPLC analysis of antiepileptics and some metabolites in plasma [15] Reagents and their preparation • Ethosuximide, primidone, phenobarbital, carbamazepine, phenytoin and carbamazepine- epoxide can be purchased from Sigma. Lamotrigine, carbamazepine-diol and 9-hydroxy- methyl-10-carbamyl acridan were reported to be obtainable from Chiba-Geigy (Basel, Switzerland) d .  ese compounds are dissolved in methanol to prepare 1 mg/mL solution. • By diluting the methanolic 1 mg/mL solution, various concentrations of some drugs in the range of 0.5–200 µg/mL are prepared for constructing each calibration curve. HPLC conditions Column: a reversed phase column a , Supelcosil LC-18 (150 × 4.6 mm i.d., particle diameter 5 µm, Supelco, Bellefonte, PA, USA). HPLC chromatogram for 8 antiepileptics spiked into human serum [11]. 1: ethosuximide (10 µg/mL, retention time 2.6 min); 2: primidone (1 µg/mL, 2.7 min); 3: phenobarbital (1 µg/mL, 4.3 min); 4: carbamazepine (1 µg/mL, 6.9 min); 5: phenytoin (1 µg/mL, 8.0 min); 6: oxazepam (1 µg/mL, 9.0 min); 7: nitrazepam (1 µg/mL, 9.6 min); 8: diazepam (1 µg/mL, 28.1 min); 9: 5-(4-methylphenyl)-5- phenyl hydantoin (IS, 1 µg/mL). ⊡ Figure 5.2 HPLC analysis of antiepileptics and some metabolites in plasma 354 Antiepileptics Mobile phase: 0.01 M potassium dihydrogenphosphate solution/methanol/acetonitrile (65:18:17, v/v) (pH 7.5); detection wavelength: 220 nm;  ow rate: 1 mL/min: column (oven) temperature: room temperature. Procedure e i. A 100-µL aliquot of plasma b and 20 µL IS solution are mixed for several seconds in a 2-mL volume microtube. ii. A 1-mL volume of diethyl ether is added to the above mixture. iii. A er shaking for 5 min, the tube is centrifuged at 1,000 g for 10 min. iv.  e organic phase (upper layer) is transferred to another clean microtube. v.  e phase is evaporated to dryness under a stream of nitrogen. vi.  e residue is dissolved in 100 µL of the mobile phase. vii. A 20-µL aliquot of the solution is injected into HPLC. viii. For constructing calibration curves, various concentrations of standard solutions are mixed with 20 µL IS solution each and processed according to the above procedure. Assessment of the method In this method, 6 kinds of antiepileptics ( ethosuximide, primidone, lamotrigine, phenobarbi- tal, phenytoin and carbamazepine) and two carbamazepine metabolites ( carbamazepine-diol and carbamazepine- epoxide) can be detected from human plasma ( > Figure 5.3).  e detec- tion limits of each drug was about 0.2 µg/mL; recovery rates were 71–104 %. Toxic and fatal concentrations in blood [21] Phenytoin: therapeutic, 7 µg/mL (10–20 µg/mL); toxic, 48 µg/mL (30–60 µg/mL); fatal, not lower than 70 µg/mL (average 94 µg/mL). Carbamazepine: therapeutic, 6.4 µg/mL (3.5–9.4 µg/mL): toxic, 10.1 µg/mL (3.2–20.6 µg/ mL); fatal, 19.6 µg/mL. Ethosuximide: therapeutic, 44 µg/mL (13–71 µg/mL); toxic, 100–200 µg/mL; fatal, 250 µg/mL. Primidone: therapeutic, 10.5 µg/mL (6–17.8 µg/mL): toxic, 20–50 µg/mL; fatal, 65 µg/mL. Diazepam: therapeutic, 0.05–2 µg/mL; toxic, 2–5 µg/mL; fatal, not lower than 5 µg/mL. Phenobarbital: therapeutic, 12.3 µg/mL (4–26.2 µg/mL); toxic, 16.7 µg/mL (3.7–90 µg/mL); fatal, 45 µg/mL (4.3–120 µg/mL). Oxazepam: therapeutic, 0.5–2 µg/mL; toxic, not lower than 2 µg/mL; fatal, not clear. Nitrazepam: therapeutic, 0.044 µg/mL (0.026–0.066 µg/mL); toxic, 0.2 µg/mL (a single oral intake); fatal, 5.2–9 µg/mL. 355 Poisoning cases Case 1 [22]: a 85-year-old female su ered from fever, muscle sti ness and itching exanthemas at home just before admission to a hospital. As her past history, she had experienced hysterec- tomy, ischemic heart disease and diabetes mellitus (type II). About one month before admis- sion, she had been diagnosed as complex partial epileptic seizure and had daily taken 300 mg phenytoin (once at night), 500 mg metformin (twice a day), 500 mg tolbutamide (twice a day), 10 mg nifedipine (twice a day) and 1 mg risperidone (once a day). She had no history of smok- ing, and had quitted drinking alcohol since the appearance of the epileptic attack. Upon medi- cal examination for admission to a hospital, she complained of her bad general conditions; her physical conditions were: body temperature 38 °C, heart beat 90/min and blood pressure 120/70 mmHg.  ere were erythematous exanthemas and the characteristic sti ness of the neck; but neither photophobia nor other pathological neurological  ndings were observed. Antibiotic, corticosteroid and antihistaminic drugs were administered intravenously. All drugs for oral intake except phenytoin were discontinued on day 3 a er admission.  e administra- tion of phenytoin was also stopped on day 7. Benzodiazepine antiepileptics were used to control the attack; the functions of the kidney, liver and heart were aggravated. She died 12 days a er admission.  e concentration of blood phenytoin at admission was 13.6 µg/mL. Case 2 [23]: a 61-year-old female had been su ering from chronic headache and frequent vertigo. As a result of clinical tests, a meningioma had been discovered in the le fronto-peri- HPLC chromatogram for 6 antiepileptics and two metabolites spiked into human plasma [15]. ES: ethosuximide (25 mg/mL, retention time 1.80 min); PR: primidone (2.5 mg/mL, 2.10 min); CBZ-D: carbamazepine-diol (1 mg/mL, 2.55 min); LTG: lamotrigine (1 mg/mL, 2.95 min); PB: phenobarbital (5 mg/mL, 3.33 min); CBZ-E: carbamazepine-epoxide (1 mg/mL, 4.48 min); IS: 9-hydroxymethyl-10-carbamyl acridan (IS, 5.92 min); PT: phenytoin (2.5 mg/mL, 9.45 min); CBZ: carbamazepine (2.5 mg/mL, 10.2 min). ⊡ Figure 5.3 Poisoning cases 356 Antiepileptics etal lobe of her brain. It had been removed by surgery without any complication. A er dis- charge from the hospital, she took 100 mg phenytoin every 8 h to prevent her from epileptic attack. Four days a er her discharge, fever and nausea appeared; on the next day (on day 5 a er discharge), itching erythematous exanthemas appeared and extended to the face, chest and extremities. Seven days a er the appearance of the exanthemas, icterus, epigestric and right upper abdominal pains, bile-containing urine and polyuria appeared. Her family doctor stopped the administration of phenytoin and prescribed an antihistaminic drug. She had a past history of allergy against lorazepam, but not history of hepatitis, blood transfusion or alcohol intake. Upon visiting a university hospital, her physical conditions were: fever 38 °C, heart beat 90/min and blood pressure 120/80 mmHg.  e erythematous exanthemas extended over almost whole skin of the body; in the neck and back, the exanthemas became desquamative. Clinical tests showed normal data except for the liver function.  e blood phenytoin concen- tration was 0.95 µg/mL (normal value 10–20 µg/mL).  e  ndings of liver biopsy showed ex- tensive damages of hepatocytes associated with disarrangement of the lobules and with in am- matory cell in ltration into the portal vein and the parenchyma. Case 3 [24]: a 18-year-old male (body weight 60 kg) had attempted suicide by ingesting phenytoin capsules (amount ingested not clear); he had no history of epilepsy. Twelve hours a er ingestion, he had been brought to a hospital in the semicomatose state. Just a er admis- sion, the blood phenytoin concentration was 45 µg/mL, but his vital signs were stable.  e gastro-lavage was performed; but no capsules or their debris could not be found in the lavage  uids.  e sounds of the intestinal peristalsis decreased slightly.  erea er, the blood phenyt- oin level increased up to 114 µg/mL on the 5th day; in the second week, the level was 105 µg/ mL.  e patient was still in the state of delirium, which was not improved. Grand mal, which seemed to be secondary to the toxic encephalopathy, appeared twice. By the end of the 2nd week, the sound of intestinal peristalsis became audible; the blood phenytoin concentration decreased to 75 µg/mL. A er 3 weeks of admission, the phenytoin could not be detected from his blood. Notes a) In many reports, reversed phase octadecyl (C 18 ) chemical-bonded silica gel columns are being used. b) Blood, serum and plasma specimens are stable at –20 °C for at least 4 weeks; they are stable at 25 °C for 24 h. c) Phenytoin has optical isomers.  eir determination method is described in the reference [6]. d) Lamotrigine and carbamazepine-diol are not obtainable in Japan; the analytical data of these compounds has been shown only as useful informations. e) For extraction of antiepileptics by solid-phase extraction, the following procedure can be recommended. i. A 5-mL volume of 20 % methanol aqueous solution and 5 mL distilled water are passed through a Sep-Pak C 18 cartridge (Waters, Milford, MA, USA) to activate it. ii. 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